Single-particle reconstruction from cryo-electron micrographs has proven to be a powerful technique for studying structure and function of biological macro-molecules. Many of these studies are not feasible by any other technique. SPIDER (System for Processing Image Data in Electron microscopy and Related fields) has been the foremost software system for carrying out such a reconstruction, especially for non-symmetrical objects. Developed in the laboratory of Dr. Joachim Frank, SPIDER software is currently providing 6.8 Angstrom reconstructions of non-symmetrical objects such as ribosomes and efforts are underway to push the resolution toward the atomic range (<3.5 A). SPIDER has been available to the electron microscopy community for more than 30 years and for almost 4 years as 'Free Open Source Software'. In 2008 alone there were 678 downloads by registered sites. The standard reference for the system has more than 800 methodological citations. Single-particle reconstruction software is unique in the scale of image sizes that it handles, the diversity of image processing techniques used, and the great computational load of many of the processing steps. Aims: 1: Increase speed/accuracy of single-particle reconstruction by implementing new alignment and back-projection methods, off-loading computations to GPUs, and adding parallel MPI I/O. 2: Develop efficient methods for handling large datasets with more than one million 500x500 pixel images, and for phase-plate imaging. 3: Further develop JWeb and SPIRE's graphical user interfaces, and better integrate them with SPIDER. 4: Collaborate with other developers to integrate outside solutions for alignment, heterogeneous samples, and interoperability into SPIDER. 5: Maintain SPIDER to take advantage of new hardware and compiler capabilities and distribute compiled, tested executables to users. PUBLIC HEALTH RELEVANCE: The SPIDER software developed and distributed by this grant is being used by a sizable community of structural biologists in research projects to increase our understanding of the structure and mechanism of action of large biological molecules and molecular assemblies. Used to analyze images from electron microscopes, SPIDER allows creation of density maps and, through fitting of X-ray structures, models of cellular objects including ribosomes, nuclear pores, molecular motors, transport molecules, viruses, and large enzymatic complexes. Such basic structural knowledge is crucial to the understanding of the processes in normal and abnormal cell behavior. As such the knowledge is directly relevant to the core mission of the NIH.